疏肝利胆汤抑制兔胆结石形成的作用及机制研究

[目的]观察疏肝利胆汤对胆石症家兔胆汁成分和胆汁成石趋势的影响。[方法]将30只家兔随机分为正常对照组、胆结石病理组、疏肝利胆汤治疗组,正常对照组每日喂普通颗粒饲料100g;胆结石病理组每日喂含1.2%高胆固醇饲料100g;疏肝利胆汤治疗组每日喂含1.2%高胆固醇饲料100g及疏肝利胆汤13ml,喂养8周,观察疏肝利胆汤对胆石症家兔胆汁成分和胆汁成石趋势的影响。[结果]疏肝利胆汤组胆汁中的总胆固醇、胆汁成石指数显著低于胆结石病理组,胆囊成石率也较结石组低,差异有统计学意义。[结论]疏肝利胆汤能够显著降低胆汁中的胆固醇含量,抑制胆石成石趋势,减少胆囊结石的发生。     

养肝利胆颗粒对胆色素结石炎症反应环节的影响

[目的]观察养肝利胆颗粒（YGLD）对豚鼠胆色素结石形成过程中炎症环节的影响。[方法]应用雄性豚鼠皮下注射林可霉素联合喂养致石饲料建立胆色素结石模型,观察YGLD对该模型成石率、胆囊容积、胆汁中黏蛋白及C反应蛋白（CRP）浓度的影响。[结果]YGLD组与模型组比较成石率显著降低（P〈0．01）,胆囊容积明显减小（P〈0．05）,胆汁中黏蛋白及CRP浓度明显降低（P〈0．05）。[结论]YGLD通过降低胆汁中黏蛋白、CRP水平来发挥逆转成石胆汁及降低胆色素结石成石率的重要作用。     

胆宁片治疗豚鼠胆色素结石的实验研究

[目的]研究胆宁片治疗豚鼠胆色素结石的作用及机制。[方法]构建豚鼠胆色素结石模型,给予胆宁片小、中、大剂量治疗,治疗后观察豚鼠成石率,并用ELISA法测定血清中IL-15、IL-1β及胆汁中IL-15含量。[结果]胆宁片中、大剂量治疗后的豚鼠成石率较模型组下降,差异有统计学意义（P〈0.01）;与模型组比较,胆宁片各剂量组血清中IL-15、IL-1β及胆汁中IL-15含量减少,差异有统计学意义（P〈0.01）,且胆宁片各剂量组间比较差异也有统计学意义（P〈0.01）。[结论]胆宁片能有效治疗豚鼠胆色素结石,其机制与减少IL-15、IL-1β含量有关。     

胆汁酸受体FXR和TGR5在溃疡性结肠炎中的表达

背景:发达国家溃疡性结肠炎(UC)的发病率高于发展中国家,可能与西化的生活方式有关,特别是高动物蛋白和低复杂碳水化合物的饮食习惯。随着高脂肪、高肉类摄入的增加,肝脏合成和分泌的胆汁酸相应增加,可能对UC的发生产生影响。目的:观察法尼醇X受体(FXR)、G蛋白偶联胆汁酸受体5(TGR5)在UC中的表达。方法:选取2013年1月—2016年6月南京医科大学附属江宁医院30例活动期UC患者,以30名健康者作为对照。采用免疫组化法检测结肠黏膜中FXR和TGR5表达。结果:与对照组相比,UC组FXR表达显著降低(4.63±2.07对6.91±2.62,P=0.00),TGR5表达无明显差异(6.70±2.90对6.11±2.44,P=0.40)。右半结肠炎患者的FXR表达显著高于左半结肠炎患者(P0.05)。结论:UC患者结肠黏膜FXR表达明显减少,在UC发病中可能起一定的作用,而TGR5在UC发病中可能无明显作用。     

黏蛋白和肠三叶因子在溃疡性结肠炎中的表达

[目的]研究溃疡性结肠炎(UC)结肠黏膜中的黏蛋白MUC2、MUC5AC以及肠三叶因子(TFF3)的表达规律及其与中医证型的关系。[方法]选择UC患者74例,其中大肠湿热证组48例,脾胃气虚证组26例,另外选10例健康体检人群为正常对照组。通过免疫组织化学染色法测定3组人群肠黏膜上皮中MUC2、MUC5AC以及TFF3的表达情况。[结果]MUC2在UC大肠湿热组、脾胃气虚组患者表达较正常对照组减少,但差异无统计学意义(P0.05)。大肠湿热组、脾胃气虚组MUC5AC的阳性表达率分别为33.3%和23.1%,正常对照组均为阴性,大肠湿热组MUC5AC阳性表达率与正常对照组相比差异有统计学意义(P0.05),而脾胃气虚组与正常对照组对比差异无统计学意义。大肠湿热组TFF3阳性表达率为79.2%,高于脾胃气虚组的53.9%,两者的阳性表达与正常对照组相比,均差异有统计学意义(P0.05)。脾胃气虚组与大肠湿热组间MUC2、MUC5AC以及TFF3的表达均差异无统计学意义(P0.05)。[结论]UC大肠湿热证MUC5AC的表达上调和TFF3的表达下调可能与UC的发生、发展有关,而且对临床诊疗、判断预后也有较大意义。     

大柴胡颗粒治疗高脂饲料致豚鼠胆囊炎的机制

目的探讨大柴胡颗粒治疗慢性胆囊炎的机制。方法将豚鼠随机分为空白对照组、模型组、低、高剂量大柴胡颗粒组(4、8 g/kg)组、消炎利胆片组(1.2 g/kg)。采用饮饲法复制胆囊炎豚鼠模型,观察大柴胡颗粒对胆囊炎豚鼠血清内皮素(ET)、一氧化氮(NO)、肿瘤坏死因子(TNF)-α含量的影响。利用免疫组化的方法,观察胆囊组织肿瘤坏死因子(TNF)-α表达情况。结果大柴胡颗粒大剂量组可显著提高胆囊炎豚鼠血清ET含量(P0.01),并可显著降低胆囊组织TNF-α表达(P0.05)。但大柴胡颗粒组对血清NO、TNF-α含量均未见明显影响。结论大柴胡颗粒对胆囊炎豚鼠的保护作用可能与提高血清ET水平及降低胆囊组织TNF-α表达有关,其他的相关机制有待进一步研究。     

溃结灵对溃疡性结肠炎大鼠结肠黏膜T L R4蛋白水平的作用

目的:观察溃疡性结肠炎(UC)大鼠结肠黏膜Toll样受体4(TLR4)的变化特点及溃结灵对TLR4的影响.方法:采用三硝基苯磺酸(TNBS)法制作UC大鼠模型,大鼠随机分为6组:正常对照组、模型对照组、溃结灵低、中、高剂量组、阳性药柳氮磺胺吡啶(SASP)组.治疗10d后处死大鼠取新鲜结肠黏膜标本提取全细胞蛋白.采用蛋白免疫印迹(Westem blot)方法对TLR4的蛋白表达水平进行检测,以β-actin作为内参,以目的蛋白与β-actin密度的比值作为目的蛋白的相对含量并进行统计学分析.结果:模型组TLR4蛋白相对表达量明显高于正常组(0.843±0.201 vs 0.472±0.072,P＜0.01);溃结灵高剂量组TLR4相对表达量明显低于模型组(0.620±0.178 vs 0.843±0.201,P＜0.05).结论:TLR4可能参与了uc大鼠的发病过程,溃结灵使TNBS法UC大鼠模型结肠黏膜TLR4的蛋白表达水平明显降低,这可能是其治疗UC作用的机制之一.     

大黄灵仙方对胆固醇结石豚鼠模型胆囊Cajal间质细胞中scf/c-kit信号通路的影响

目的 观察在大黄灵仙方的调控下干细胞生长因子(scf)、酪氨酸激酶受体(c-kit)的表达水平，探讨其影响胆囊动力学改变的可能机制，为大黄灵仙方预防胆石症的发生及复发提供理论依据。方法 将45只SPF级健康雄性豚鼠随机分为正常组、模型组、中药组，其中正常组予正常饲料喂养，模型组、中药组予高脂致石饲料喂养，喂养8周后分别从各组中随机抽取5只豚鼠，肉眼观察下结石形成超过4只，则判断为模型建立成功。造模成功后中药组予大黄灵仙方灌胃，模型组予等体积生理盐水灌胃，连续灌胃给药8周后取豚鼠胆囊组织,HE染色观察观察胆囊组织的病理学改变,Western blot测定胆囊组织中TNFα的表达水平，免疫组化测定胆囊平滑肌组织中scf、c-kit的蛋白表达水平。符合正态分布的计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD多重比较法。结果 HE染色显示模型组胆囊组织炎症明显，中药组胆囊组织内炎症程度较模型组明显减轻。Western blot结果表明在模型组中，胆囊组织中TNFα的表达水平最高，中药组次之，正常组最低(P值均<0.05);免疫组化结果表明豚鼠胆囊平滑肌组织中scf、c-k...     

Toll样受体4单克隆抗体干预小鼠实验性溃疡性结肠炎对信号转导通路的影响

目的 探讨Toll样受体4单克隆抗体(Toll like receptor 4 monoclonal antibodies,TLR4mAb)干预葡聚糖硫酸钠(DSS)诱导溃疡性结肠炎(UC)小鼠肠黏膜中TLR4-P38丝裂原活化蛋白激酶(MAPK)-c-fos/c-jun信号转导通路的影响情况.方法 30只BALB/C小鼠分为对照组、模型组以及低、中、高剂量干预组.对照组小鼠饮用蒸馏水7 d;其他四组小鼠饮用5%DSS溶液7 d建立急性期UC模型.造模同时,干预组小鼠分别以低(2 μg)、中(10 μg)、高剂量(20 μg)TLR4mAb腹腔注射,共4次,7 d后处死小鼠,观察疾病活动指数(DAI)、结肠组织病理学评分(HPS).RT-PCR法检测各组肠黏膜TLR4的mRNA表达,Western印迹法测定各组肠黏膜P38MAPK、磷酸化P38MAPK、c-fos、c-jun的蛋白表达.结果 模型组TLR4 mRNA表达明显高于对照组(P＜0.01).与模型组相比,低、中、高剂量干预组DAI指标和HPS指标均有不同程度缓解.P38MAPK及c-jun蛋白表达在低、中、高剂量干预组呈逐步递减,且均较模型组有明显下降(P＜0.01).与模型组相比,低、中、高剂量干预组小鼠肠黏膜磷酸化P38MAPK蛋白及c-fos蛋白表达差异均无统计学意义(P＞0.05).结论 早期应用TLR4mAb对DSS诱导小鼠急性期UC有干预作用,其作用机制可能与抑制TLR4--P38MAPK--c-jun信息转导通路有关.     

儿茶素调控MAPK通路改善幼鼠过敏性气道炎症反应的机制研究

目的探索儿茶素调控丝裂原活化蛋白激酶(MAPK)通路改善幼鼠过敏性气道炎症反应的机制。方法选取SPF级BALB/c纯系小鼠50只,根据随机数字表法分为模型组、正常组、阳性对照组、儿茶素低剂量组及儿茶素高剂量组,每组各10只。除正常组外,其他各组小鼠制备过敏性哮喘模型,成功造模后阳性对照组小鼠灌胃剂量为0.5 mg/kg的地塞米松药液,儿茶素低剂量组及儿茶素高剂量组分别灌胃剂量为1 mg/kg、2 mg/kg儿茶素药液,模型组和正常组灌胃等剂量生理盐水,1次/d。观察小鼠支气管肺泡灌洗液(BALF)内分类细胞、总细胞计数及白细胞介素13(IL-13)、IL-5、IL-4水平,检测小鼠肺组织内p-p38MAPK、ERK、p-ERK、p38MAPK蛋白表达及p38MAPK、ERK mRNA表达。结果与正常组比较,模型组小鼠BALF内IL-13、IL-5、IL-4水平均升高。与模型组比较,阳性对照组、儿茶素低剂量组及儿茶素高剂量组小鼠BALF内IL-13、IL-5、IL-4水平升高降低,差异均有统计学意义(P值均<0.05)。与正常组比较,模型组小鼠肺组织内p-p38MAPK、p-ERK蛋白表达升高。与模型组比较,阳性对照组、儿茶素低剂量组及儿茶素高剂量组小鼠肺组织内p-p38MAPK、p-ERK蛋白表达均降低,差异均有统计学意义(P值均<0.05)。与正常组比较,模型组小鼠肺组织内p38MAPK、ERK mRNA表达均上升,差异均有统计学意义(P值均<0.05)。与模型组比较,阳性对照组、儿茶素低剂量组及儿茶素高剂量组小鼠肺组织内p38MAPK、ERK mRNA表达均降低,差异均有统计学意义(P值均<0.05)。结论儿茶素可使过敏性哮喘小鼠气道内炎症反应有效改善,其作用机制可能和抑制ERK/MAPK信号路径激活有联系。     

中药胆宁片治疗胆囊胆固醇结石作用机制的实验研究

[目的]探讨胆宁片对胆固醇结石豚鼠胆汁中黏蛋白及血清IL-2水平的影响.[方法]雌性豚鼠60只,体重（300±20）g,随机分为空白组、模型组、胆宁片组和熊脱氧胆酸组,每组15只;除空白组外,采用“高胆固醇致石食饵诱发法”建立豚鼠胆固醇结石模型,并于造模成功后分别对各治疗组予药物干预,胆宁片组灌服胆宁混悬液0.52 g· kg-1·d-1,熊脱氧胆酸组灌服熊脱氧胆酸混悬液0.05 g·kg-1·d-1,模型组与空白组均灌服等容量的生理盐水,8周后观察各组豚鼠一般情况、胆汁中黏蛋白及血清IL-2水平.[结果]胆宁片可显著降低豚鼠胆固醇结石成石率,并能显著降低胆囊黏蛋白及血清IL-2水平（P＜0.05,P＜0.01）.[结论]胆宁片能直接调节胆囊黏蛋白等相关促成核因子,同时能通过调节血清中IL-2间接调控胆囊黏蛋白水平,从而调控成核过程,对胆囊胆固醇结石有一定的治疗作用.     

Effect of age and sensitivity to cholecystokinin on gallstone formation in the guinea pig

The purpose of this study was to evaluate the effect of age and the role of cholecystokinin therapy on gallstone formation in guinea pigs. Guinea pigs (31 1-mo-old, 31 1-yr-old, and 23 3-yr-old) were placed on a cholelithogenic diet for 2 wk while another 10 guinea pigs of each age group remained on regular chow. Half of each group received a daily injection of cholecystokinin (0.5 nmol/kg). After 2 wk, guinea pigs were killed and the gallbladders were examined for gallstones. The concentrations of bile constituents were determined. The prevalence of gallstones was: 1-mo-old, control 0 out of 16, cholecystokinin 1 out of 15; 1-yr-old, control 3 out of 14, cholecystokinin 5 out of 16; 3-yr-old, control 10 out of 11, cholecystokinin 3 out of 8. Gallstone formation was significantly greater in 3-yr-old controls than in the two younger control groups, and cholecystokinin treatment significantly reduced the incidence of gallstones to near the level seen in younger guinea pigs. In the two younger age groups (but not in the 3-yr-old group), the cholelithogenic diet significantly reduced the concentration of bile salts in bile below that of guinea pigs on a normal diet. The cholelithogenic diet and treatment with cholecystokinin did not alter the relative compositions of bile lipids from that of guinea pigs on a normal diet in any of the three ages studied. In the second experiment we measured gallbladder emptying in response to exogenous infusion of cholecystokinin-8 (100 fmol/kg/h-100 nmol/kg/h) in the same three age groups of guinea pigs in vivo that had been maintained on regular chow. There was no difference in cholecystokinin sensitivity between the two younger age groups, but both were significantly more sensitive to cholecystokinin than the 3-yr-old guinea pigs in rate of gallbladder emptying in the dose range 1 pmol/kg/h-1 nmol/kg/h. We conclude that a major factor in the increased incidence of gallstone formation in the aged guinea pig gallstone model is decreased gallbladder emptying due to decreased gallbladder sensitivity to cholecystokinin.     

胆胃舒颗粒对胆囊血管活性肠肽受体基因表达的影响

[目的]观察胆胃舒颗粒对胆囊血管活性肠肽(vasoactive intestinal peptide,VIP)受体基因表达的影响及预防胆囊结石的作用.[方法]雄性豚鼠90只,随机分为3组,每组30只,空白组喂养普通饲料40 g/(d·只);模型组喂养胆固醇结石诱石饲料40 g/(d·只);治疗组喂养胆固醇诱石饲料40 g/(d·只),加胆胃舒颗粒溶液1.5ml(含300mg胆胃舒颗粒)灌胃.实验2个月后观察3组胆囊结石情况、胆囊收缩功能及胆囊壁VIP受体基因表达.[结果]胆囊结石形成率:空白组3.33％(1/30),模型组92.59％(25/27),治疗组10.71％(3/28);胆囊收缩功能:空白组胆囊收缩率为(66.83± 5.34)％,模型组(43.06±4.27)％,治疗组(67.93±6.82)％;胆囊壁VIP受体基因表达:空白组0.30±0.07,模型组0.45±0.12,治疗组0.33±0.06.差异均有统计学意义(P＜0.05).[结论]胆胃舒颗粒可降低胆囊结石的形成,其作用机制可能通过降低胆囊壁VIP受体基因表达,从而加强胆囊收缩功能,促使胆汁排泄,防止胆囊结石的发生.     

硫化氢/胱硫醚-γ-裂解酶在胆管梗阻致急性胆囊炎中的表达

目的探讨硫化氢/胱硫醚-γ-裂解酶(H2S/CSE)体系在胆管梗阻致急性非结石性胆囊炎(AAC)中的表达。方法健康成年豚鼠30只,胆总管结扎(BDL)方法构建豚鼠AAC模型。随机分为3组,即正常组、假手术对照(Sham)组和胆总管结扎48 h(BDL-48 h)组。HE染色后光镜下观察各组胆囊病理改变;检测胆囊组织中CSE活性;免疫组化SP法观察各组胆囊组织中CSE的表达;免疫印迹法分析各组胆囊CSE的表达水平。结果正常组和Sham组未见明显炎症反应,与正常组、Sham组相比,BDL-48 h组胆囊组织病理学评分差异有统计学意义(P0.05)。各组胆囊组织中CSE呈阳性表达,而BDL-48 h组的CSE活性、CSE蛋白表达均较正常组和Sham组显著增高(P0.05)。结论胆囊组织存在CSE,H2S/CSE体系可能参与了AAC的病理过程。     

Can pigment gallstones be induced by biliary stricture and prevented by medicine in Guinea pigs？

AIM: To determine the relationship between biliary stricture and pigment gallstone formation, and the prevention of pigment gallstones with medicine. METHODS: One hundred and eighteen male guinea pigs were randomly divided into four groups: stricture group (S, n = 30) underwent partial ligation of common bile duct, and fed on regular chow; S plus medicine group (S+M, n = 27) underwent the same operation but fed on medicinal chow (0.3 g chenodeoxycholic acid, 0.5 g glucurolactone, and 0.5 g aspirin were mixed up in 1.2 kg regular chow); medicinal control group (C+M, n = 30) was free of operation, and fed on medicinal chow; and control group (C, n = 31) was free of operation and fed on regular chow. One week later, laparotomy was performed, and the bile of gallbladder was collected, measured, and cultured. RESULTS: Gallstones were identif ied. Pigment gallstones were induced by biliary stricture in 95% (22/23) of S group. In the S+M group, the incidence of gallstone was reduced to 55% (11/20, vs S group, P < 0.01). The changes of indirect bilirubin and ionized calcium in the bile were consistent with gallstone incidences. CONCLUSION: Biliary stricture can cause pigment gallstone formation in guinea pigs, and the medicines used can lower the incidence of gallstones. The bilirubin and ionized calcium play important roles in pigment gallstone formation.     

Effects of sphincter of Oddi motility on the formation of cholesterol gallstones

AIM: To investigate the mechanisms and effects of sphincter of Oddi (SO) motility on cholesterol gallbladder stone formation in guinea pigs.METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups, the control group (n = 10) and the cholesterol gallstone group (n = 24), which was sequentially divided into four subgroups with six guinea pigs each according to time of sacrifice. The guinea pigs in the cholesterol gallstone group were fed a cholesterol lithogenic diet and sacrificed after 3, 6, 9, and 12 wk. SO manometry and recording of myoelectric activity were obtained by a multifunctional physiograph at each stage. Cholecystokinin-A receptor (CCKAR) expression levels in SO smooth muscle were detected by quantitative real-time PCR (qRT-PCR) and serum vasoactive intestinal peptide (VIP), gastrin, and cholecystokinin octapeptide (CCK-8) were detected by enzyme-linked immunosorbent assay at each stage in the process of cholesterol gallstone formation.RESULTS: The gallstone formation rate was 0%, 0%, 16.7%, and 83.3% in the 3, 6, 9, and 12 wk groups, respectively. The frequency of myoelectric activity in the 9 wk group, the amplitude of myoelectric activity in the 9 and 12 wk groups, and the amplitude and the frequency of SO in the 9 wk group were all significantly decreased compared to the control group. The SO basal pressure and common bile duct pressure increased markedly in the 12 wk group, and the CCKAR expression levels increased in the 6 and 12 wk groups compared to the control group. Serum VIP was elevated significantly in the 9 and 12 wk groups and gastrin decreased significantly in the 3 and 9 wk groups. There was no difference in serum CCK-8 between the groups.CONCLUSION: A cholesterol gallstone-causing diet can induce SO dysfunction. The increasing tension of the SO along with its decreasing activity may play an important role in cholesterol gallstone formation. Expression changes of CCKAR in SO smooth muscle and serum VIP and CCK-8 may be important causes of SO dysfunction.     

无症状胆石症患者胆囊运动功能及阿期匹林的作用

为研究无症状胆囊结石患者胆囊动力变化及阿斯匹林的影响． 采用腹部超声法观察４３例无症状胆囊 结石（ＡＧ）患者和 ２０名健康对照服用阿斯匹林（０．３／日．６天）前后胆囊容量的变化，结果：（１）ＡＧ组空腹胆囊容积 （ＦＧＶ）和剩余胆囊容积（ＲＧＶ）明显增大（Ｐ＜０．０１），胆羹排空率（ＧＥＲ）明显减少（Ｐ＜０．０１）．无论在快速排空或 缓慢排空期．ＡＧ组胆囊排空功能均有明显损害；（２）服用阿斯匹林后未观察到正常组（ＨＳ）和ＡＧ组织胆囊排空率 的改善。提示．ＡＧ组存在胆囊动力障碍，胆囊排空功能障碍可能是胆囊结石成因之一：前列腺素抑制剂阿斯匹林 对胆囊的动力障碍无改善。     

Early stages of gallstone formation in guinea pig are associated with decreased biliary sensitivity to cholecystokinin

The purpose of this study was to measure differences in gallbladder sensitivity to cholecystokinin (CCK)in vivo during the early stages of gallstone formation and to correlate these findings to gallbladder CCK receptors. Guinea pigs were placed on either a normal diet or a two-week cholelithogenic diet, after which gallbladder emptying pressure to exogenously administered CCK was measuredin vivo, according to the presence or absence of gallstones. At all doses of CCK tested (except 10^–10 mol/kg), the gallbladder response to CCK of guinea pigs that did not develop gallstones (on the cholelithogenic diet) was more sensitive than that of guinea pigs that did develop gallstones. Neither group was different from guinea pigs on a normal diet. In a second experiment, CCK receptors were measured on gallbladder muscularis from guinea pigs after two weeks on the same diet as in the first experiment. Those guinea pigs that did not develop gallstones had greater concentrations of CCK receptors (149±9 fmol/mg protein) than those that did develop gallstones (70±23 fmol/mg protein). Neither group was different from normal diet guinea pigs (119±57 fmol/mg protein). At the time point measured, there were no differences in the lipid chemistry, or protein concentrations of gallbladder bile between the guinea pigs on the cholelithogenic diet that did or did not develop gallstones, or those on normal guinea pig chow. We conclude that the early stages of gallstone formation in guinea pigs are associated with decreased gallbladder sensitivity to CCK and that this change may be due to a lower concentration of CCK receptors on the gallbladder smooth muscle.Supported by the Wellcome Foundation, Ethicon Foundation, and British Digestive FoundationSupported by grants from the National Institutes of Health (5R37 DK 15241-19, P01 DK 35608, and CA 38657)